In many research, industrial and clinical laboratories there is a growing need for indentification of a single protein present in a complex protein mixture. This is most often achieved by one or two-dimensional electrophoretic separation of proteins followed by Western blotting. Here Western blotting includes transfer of proteins to a suitable membrane, reaction with antibodies specific for one particular protein and visualization of the antibodies bound to that protein. The visualization of antibodies is done in an indirect way, that is through a signal generated by a label attached to the antibody molecule. The label is very often an enzyme but other molecules producing a suitable signal, such as fluorescent dyes or radioactive compounds, are also in use. There are several ways to couple a label to an antibody, and the properties of the resulting conjugate are greatly dependent on the coupling method used for its preparation (reference 1).
In addition to detection of a single protein, it is sometimes necessary to identify a certain class of proteins. This can be achieved through a common determinant of these proteins. For example, glycoproteins can be detected through the carbohydrate chains that are covalently linked to the protein part of the molecule. Thus, the intact carbohydrate chains can be detected by means of proteins, such as lectins, glycosidases or glycosyltransferases, that recognize those monosaccharides that are present in the glycan chains (reference 2). A more general approach involves a modification of the carbohydrate chains followed by their detection through a group introduced by the modification. It is obvious that such a group must not be present in an intact protein or glycoprotein molecule. Aldehyde groups are easily and conveniently generated in the sugar chains, either chemically or enzymatically, and then detected by means of a hydrazide reagent and a suitable label (reference 2).
Antibodies, that is immunoglobulins, are also glycoproteins. Therefore, their carbohydrate chains can be used to introduce a label. This approach is very attractive because the carbohydrate chains of antibodies are apparently not directly involved in the binding of an antigen. Several reports have appeared describing preparation of such conjugates (references 3 and 4). In one report (references 5) it was claimed that an enzyme was coupled to the sugar portion of the antibody, but the procedure described was later found unsatisfactory (reference 6). We have found that aldehyde groups introduced into IgG can be used to cross-like the carbohydrate chains present on each of the two IgG heavy chains (reference 7). The cross-linking reaction by dihydrazide molecules was much more favorable than the reaction of dihydrazides through only one hydrazide group. This finding has indicated that it is difficult to introduce free hydrazide groups into IgG by using a dihydrazide. As demonstrated in this invention, that can be readily achieved by polymeric hydrazides which therefore serve as a bridge to the label.